Solanum malmeanum, a promising wild relative for potato breeding

Crop wild relatives are gaining increasing attention. Their use in plant breeding is essential to broaden the genetic basis of crops as well as to meet industrial demands, for global food security and sustainable production. Solanum malmeanum (Solanum sect. Petota, Solanaceae) is a wild relative of potatoes (S. tuberosum) from Southern South America, occurring in Argentina, Brazil, Paraguay and Uruguay. This wild potato has been largely mistaken for or historically considered as conspecific with S. commersonii. Recently, it was reinstated at the species level. Retrieving information on its traits and applied uses is challenging, because the species name has not always been applied correctly and also because species circumscriptions and morphological criteria applied to recognize it have not been consistent. To overcome these difficulties, we performed a thorough literature reference survey, herbaria specimens’ identification revision and genebank database queries to review and update the information available on this potato wild relative, contributing to an increase in research on it to fully understand and explore its potential for potato breeding. Scarce studies have been carried out concerning its reproductive biology, resistance against pests and diseases as well as tolerance to abiotic stresses and evaluation of quality traits. The scattered information available makes it less represented in genebanks and genetic studies are missing. We compile, update and present available information for S. malmeanum on taxonomy, geographical distribution, ecology, reproductive biology, relationship with its closest relatives, biotic and abiotic stresses resistance and quality traits and discuss ways to overcome sexual barriers of hybridization and future perspectives for its use in potato breeding. As a final remark, we highlight that this species’ potential uses have been neglected and must be unlocked. Thus, further studies on morphological and genetic variability with molecular tools are fundamental for an efficient conservation and applied use of this promising genetic resource.


Introduction
Potato (Solanum tuberosum L.) is the third most important food crop in terms of global food security, after rice and wheat and the most important non-grain food crop (Birch et al., 2012). It is a prominent source of calories, fibers, proteins, mineral micronutrients, vitamins B6 and C, potassium, and antioxidant compounds, and can pack all those nutrients while using less area (m²) than any other crop (Birch et al., 2012;Devaux et al., 2014). Conventional potato cultivars are bred at the tetraploid level, have high heterozygosity, reproduce vegetatively and have less genetic variation than their wild relatives (Fu et al., 2009;Hardigan et al., 2015;Kolech et al., 2016;Hardigan et al., 2017;Duan et al., 2019;Drapal et al., 2020). Climate change and the dispersion of pests and new races of pathogens are currently a matter of concern among potato breeders and farmers. The outbreaks caused by new isolates of the late blight oomycete Phytophthora infestans (Mont.) de Bary in Europe and the Americas (Santana et al., 2013;Göre et al., 2019;Maurice et al., 2019), or by the new sequevars of bacterial wilt Ralstonia solanacearum found in Brazil, Peru and Iran (Gutarra et al., 2017;Santiago et al., 2017;Sedighian et al., 2020) have the potential to destroy potato fields. Lack of resistance of the current potato cultivars to the main diseases that affect the crop makes it one of the most dependent crops on agrochemical applications (Vleeshouwers et al., 2011). This scenario requires genetic variability for the continuous process of crop improvement to face biotic stressors and climate change, while increasing productivity to ensure global food security.
Crop Wild Relatives (CWR) are a reservoir of valuable genetic diversity for crop improvement, especially those genes that confer resistance to pests and diseases, tolerance to abiotic stressors and climate change scenarios, as well as providing nutritional compounds (Maxted et al., 2006;Jansky et al., 2013;Zhang et al., 2017;Pradheep et al., 2019;Perrino and Perrino, 2020). CWR contain pharmaceutical and industrial properties and can potentially contribute for a sustainable and smarter agroecosystem way (Jansky et al., 2013;Dempewolf et al., 2014;Warschefsky et al., 2014).
The cultivated potato has more than 100 wild relative species (Solanum sect. Petota, Solanaceae) (Douches et al., 1989;Hawkes, 1990;Pavek and Corsini, 2001;Spooner et al., 2014;Hardigan et al., 2017). Potato wild relatives (PWR) are widely distributed across the Americas, from the geographical and ecological points of view, ranging from Southwestern United States (38°N), through the dry areas of Mexico, until the adjacent mainland areas of Chile (41°S) in the Southern Cone of South America, at elevation ranging from sea level up to 4,500 m (Hijmans and Spooner, 2001;Spooner et al., 2004;Spooner et al., 2014;Spooner et al., 2016;Spooner et al., 2019). About 70% of these species are diploids while autopolyploids and allopolyploids go up to 6x chromosome numbers (x=12). PWR possess a common (basic) A genome, which, modified to different degrees, originated four additional genomes: B, C, D and E (Matsubayashi, 1991). Furthermore, the differentiation in the basic A genome is assumed to play a minor role as an isolation mechanism (Camadro et al., 2004).
To guide potato breeders in the efficient use of PWR according to their gene pool level, Spooner and collaborators (2014) proposed five crossability groups, based on the Endosperm Balance Number (EBN) along the ploidy, and self-compatible/self-incompatible systems. Diploid potato species (1EBN and 2EBN) are mostly selfincompatible (Cipar et al., 1964;Spooner et al., 2014). All 4EBN potato species are self-compatible due to a lack of pre-zygotic gametophytic barriers (Spooner et al., 2014). A self-compatible plant of S. chacoense was found (Hanneman, 1985) and the responsible S-loci-inhibitor (Sli) gene was identified and mapped to chromosome 12 (Hosaka and Hanneman, 1998a;b). The EBN postzygotic barrier of hybridization acts at the embryo-endosperm development level, leading to aberrations in chromosome pairing and is observed through embryonic abortion, male sterility and breakdown of F1 or F2 hybrid offspring (Camadro et al., 2004;Camadro et al., 2012). According to Johnston et al. (1980), this post-zygotic barrier is explained under the hypothesis of the Endosperm Balance Number (EBN), in which Solanum sect. Petota species have a true ploidy number (determined by their actual number of chromosomes) and an "effective" ploidy generated by the action of hypothetical genetic factors (called EBN) (Johnston et al., 1980). The EBN system requires a 2:1 maternal to paternal EBN dosage for a normal endosperm development, which could predict the success of hybridization of some specific crosses. EBN is under genetic control (Ehlenfeldt and Hanneman, 1988;Camadro and Mansuelli, 1995;Johnston and Hanneman, 1996) and its values have been determined empirically for most PWR species by crossing each one with a standard species of known EBN (Hanneman, 1994;Mansuelli and Camadro, 1997). Due to the strong post-zygotic barrier of EBN differences, 2x (1EBN) species are sexually isolated from 2x (2EBN) and both 4x (4EBN) (C am a d r o a n d P e l o q u i n , 1 9 8 1 ; Hanneman, 1994).
Due to a changing taxonomic history, several morphological shared traits and geographical distribution patterns that greatly overlap, S. malmeanum has been largely confused or historically considered as conspecific with S. commersonii. Nevertheless, it was reinstated at the species level (Spooner et al., 2016). Due to the instability of the cohesive application of this species name and discordant species circumscriptions and morphological borders applied to recognize it along the taxonomic history, retrieving information on its traits and applied uses is tricky. To overcome these difficulties, we performed a thorough literature review, genebank database queries and a revision of herbaria voucher specimens cited in published studies to update the information available on this wild potato relative, aiming to broadening the scientific knowledge on the species to fully understand and explore its potential for potato breeding. Thus, our study aims to compile, synthetize and update all the scattered information about reproductive biology and cytogenetics, taxonomic treatments and geographical data, biotic and abiotic resistance of S. malmeanum, providing it in an elucidative and comprehensive way.

Species circumscription
Our circumscription of S. malmeanum is based on the species original publication by Bitter (1913), the revised taxonomic treatment of Spooner et al. (2016);Nicolao (2021) and complementary observations from characterized genebank accessions available at database AleloVegetal (https://av.cenargen.embrapa.br/avconsulta/ Home/index.do). When the original data discussed in this review was formerly published as S. commersonii, but the cited accession or specimen corresponds to S. malmeanum, we added the remark "as S. commersonii" right after.

Literature consulted
We reviewed the monographs for taxonomic treatments of tuberbearing Solanum species conducted by Bitter (1913); Correll (1962); Hawkes and Hjerting (1969), and Spooner et al. (2016), as well as regional taxonomic studies for Argentina by Matesevach and Barbosa (2005), South of Brazil by Mentz and Oliveira (2004)

Taxonomic and geographical distribution data
To update the taxonomic and geographic data for S. malmeanum, we reviewed, either through specimens loans or online, the label data of specimens from the herbaria B, BAL, BHCB, BM, CEN, ECT, F, ICN, K, MVFA, MVJB, MVM, MO, NY, P, R, RB, S, SPF, U, and US (acronyms according to Thiers 2022). Georeferenced records were extracted from the online platform databases Global Biodiversity Information Facility, speciesLink. Duplicate records were removed. Manual checking and correction of wrong or doubtful coordinates were carried out in GEOlocate (http://www.geo-locate.org/), Google Earth and Google Maps. The occurrence data were plotted on a map rendered in QGIS (2022).

Genebank databases search
To assess the representativeness of S. malmeanum in the germplasm banks we explored the online databases AleloVegetal (2022) by the Brazilian Agricultural Research Corporation (EMBRAPA) and Genesys (2022), a Global Portal on Plant Genetic Resources for Food and Agriculture (PGRFA). We also accessed offline data from genebank curators from Instituto Nacional de Tecnologıá Agropecuaŕia (INTA) of Argentina and Instituto Nacional de Investigacioń Agropecuaria (INIA) of Uruguay. Thus, passport information of Solanum malmeanum from INIA and INTA were obtained personally because they host critical collections of the focused species, although their data are not available at Genesys.

Agronomical traits
To compile the agronomical traits (i.e., biotic resistance, abiotic tolerance levels and quality traits), we explored the online databases Genesys (2022) and literature cited accordingly.

Criteria for updating taxonomic information
We checked and updated the taxonomic determinations of retrieved records of S. commersonii and S. malmeanum for taxonomic accuracy through the revision of voucher specimens deposited in herbaria or other verifiable sources as genebank pictures of the plants under cultivation. The confirmation, updating or corrections of identifications were possible when the studied specimens matched the current circumscription of S. malmeanum based on the observation of diagnostic morphological features present in the physical specimens or in the high-quality digital images retrieved. Accessions cited in previously published works as "S. commersonii" and "S. commersonii subsp. malmeanum" were checked by its code accession at the Germplasm Resources Information Network (GRIN Taxonomy, 2022), to confirm their correct taxonomic identification.

Results
3.1 Taxonomic history, habitat and geographical distribution Bitter (1913), when describing S. malmeanum as a new species to science, emphasized that it can be confused with S. commersonii due to their similar habit. Later, Correll (1962) downgrades S. malmeanum to a form of S. commersonii because he considers it could represent a result of hybridization between S. commersonii and S. chacoense stating that "Solanum form malmeanum" differs from the typical form of S. commersonii because the former contains 4-5 lateral leaflets pairs and two or more lateral interstitial segments while the typical form of S. commersonii contains 2-5 lateral leaflet segments pairs, without or rarely bearing lateral interstitial segments pairs. Another difference on flower characteristic reported by Correll (1962) was that S. malmeanum has its corolla narrowly and deeply lobed, often to near the base than S. commersonii. Hawkes and Hjerting (1969) states that Correll was mistaken in classifying S. malmeanum as a form of S. commersonii because they consider it consists of an assemblage of biotypes with a distinct geographical distribution which are adapted to a habitat range different from that of the typical S. commersonii. According to them, S. malmeanum has lateral leaflet segments generally decreasing gradually to the leaf base and are narrowly decurrent, being normally petiolulate, while the inflorescence peduncle is unbranched or has the branches not markedly contracted with pedicels articulation median to higher than in S. commersonii, and corollas are always white and the lobes about as long as broad.
Conversely, S. commersonii shows lateral leaflet segments decreasing rapidly in size towards the base of the leaf, being often markedly decurrent and normally sessile, while the inflorescence peduncle branches and is somewhat contracted with pedicels articulation lower to median, and corollas are generally tinged with purple and have the lobes about 1 and a ½ time as long as broad or even longer than in S. malmeanum. The two authors reinforced that the two taxa could almost be considered to differ from each other at the species level.
The last comprehensive taxonomic treatment including S. malmeanum was by Spooner and collaborators (2016). According to them, S. malmeanum can be differentiated from S. commersonii by its generally subequal uppermost lateral leaflets that do not decrease rapidly in size towards the leaf base, by its generally petiolulate and larger lateral interstitial segments and by its white corollas. The corollas are almost white, although in a very few cases they have observed a small brushstroke on the abaxial midribs of the corolla lobes very different from the characteristic dark stripes along the abaxial midribs of the corolla lobes in flowers of S. commersonii. Maybe, these specimens could represent hybrids between S. malmeanum and S. commersonii.
Solanum malmeanum is native to the Southern Cone of South America, in Argentina, Brazil, Paraguay and Uruguay. It is found in diverse environments such as shady forests, grasslands, damp pastures, roadsides, stream sides, as well as invasive at crop cultivations, including potato fields (Hawkes and Hjerting, 1969). In Argentina it is found in the Eastern Subtropical Biogeographic Province at grasslands or savannas and the Mixed Forest Biogeographic Districts, Chaco Biogeographic Province by Eastern Chaco District (more humid eastern parts) and along the Espiñal Biogeographic Province by Santa Féand Entre Rıós (Hawkes and Hjerting, 1969). In Brazil, it occurs in the Araucaria angustifolia subtropical Mixed Forests of the Paranense Biogeographic Province, with high annual rainfall, as well as in the Pampean Biogeographic Province with a dominant vegetation of grasses and bushes (Hawkes and Hjerting, 1969). In Paraguay, it spreads through the Mixed Forest Biogeographic District into the wetter parts of the Chaco Biogeographic Province and the Chaqueño Biogeographic Domain that are characterized by xerophytic vegetation (Hawkes and Hjerting, 1969). In Uruguay it is found mainly in the so-called 'Galerıá Uruguayense', that comprises the forests along the Uruguay river basin, belonging to the Mixed Forest Biogeographic District of the Eastern Subtropical Biogeographic Province and to the Pampean Biogeographic Province, with a dominant vegetation of grasses and bushes (Hawkes and Hjerting, 1969;Morrone et al.,2014;Spooner et al., 2016). Records found in the surveyed herbaria and genebanks range from sea level up to 765 m of elevation (AleloVegetal, 2022; Global Biodiversity Information Facility, 2022).

Herbarium representativeness
A total of 281 records for S. malmeanum specimens are preserved in many herbaria, of which 197 with geographical coordinates were plotted ( Figure 1).

Genebank representativeness
Currently, S. malmeanum is represented in genebanks by a total of 89 accessions worldwide (Table 1)

Genetic variability characterization
Although there are only a few studies on genetic variability which include accessions from Solanum malmeanum, in general, the results point at a close relationship with S. commersonii but allow to distinguish both species from each other and from S. chacoense (Lester, 1965;Jacobs et al., 2008;Siri et al., 2009). Through immune-electrophoretic analysis, one shared tuber antigen found in S. malmeanum and S. commersonii was absent from S. chacoense, pointing to a closer relationship between these first two species (Lester, 1965).
In order to analyze genetic variability within the species and its relationship with closely related species, molecular markers have been applied among different accessions from S. malmeanum across its distribution. Partial sequences of a new retrotransposon Tnt1 family member, Retrosol, were reported in several Solanum sect. Petota species, including S. malmeanum (Manetti et al., 2007). The accession BAL80009 presented three different fragment sequences of Tnt1 with an average of 423+/-(5) base pairs (bp) and 0.093 pJC nucleotide diversity with Jukes and Cantor (1969) corrections.
Phylogenetic analyses using AFLP markers confirmed that S. malmeanum can be genetically separated from S. chacoense and S. Geographical distribution of Solanum malmeanum (Solanaceae) in Southern South America. Occurrence points are represented by red circles for herbarium specimens and blue triangles for accessions in genebanks.   commersonii, because two of 12 accessions of S. commersonii that were allocated in the same group with seven S. malmeanum accessions (Jacobs et al., 2008), were later confirmed to in fact belong to S. malmeanum (CGN 22351, GLKS 35340). Siri et al. (2009) assessed the genetic diversity of S. commersonii and S. malmeanum from different geographical areas of Uruguay by using different PCR-based markers (three primers for AFLP, 10 primers for RAPD, and four primers for SSR). They found a high consistency of clustering with the geographic origin of accessions, with S. malmeanum (cluster B) from the north of Uruguay and S. commersonii (cluster A) from the south, previously characterized in morphology (see Siri et al., 2009). When associated with morphological features, the PCR-based markers were able to discriminate S. malmeanum from S. commersonii. Few studies on the geographic distribution of genetic diversity for S. commersonii and S. malmeanum are available. In addition, the entire potential area in which both taxa are geographically distributed has not yet been explored, and further studies are needed for a complete and more accurate coverage of the geographic distribution.
3.6 Solanum malmeanum as a source of traits for the generation of new potato cultivars 3.6.1 Frost and heat tolerance Solanum malmeanum is particularly interesting due to its freezing tolerance and great capacity to cold acclimate (i.e., increase cold tolerance after exposure to low, non-freezing temperatures) (Hawkes, 1958;Hudson, 1961;Ross and Rowe, 1965;Hawkes and Hjerting, 1969;National Plant Germplasm System, USDA, 2022;Genesys, 2022). Twenty-three accessions were reported to be frost resistant and one accession is also reported to be moderately resistant to heat stress (Table 3). It has been reported to be capable of surviving at extremely low temperatures (-0.55°C to -5°C), with none or a relatively small percentage (0-20%) of the leaf area damaged (Ross and Rowe, 1965;Hawkes and Hjerting, 1969;Vega and Bamberg, 1995). Earlier observations reported that wild potatoes that have a rosette habit, such as S. malmeanum, generally are more highly resistant to cold and frost (Firbas and Ross, 1962). Because most potato cultivars are sensitive to low temperatures and are unable to cold acclimate at temperatures lower than -3°C (Chen and Li, 1980), great efforts are carried out to find sources of tolerance and to introgress this trait into cultivated potato. A Solanum malmeanum freezing tolerant accession (MLM266-2) was fused with freezing susceptible dihaploid S. tuberosum (AC142) by somatic hybridization. Shoots were regenerated from calli formed, excised and cultured on MS medium (Tu et al., 2021). Somatic hybrids submitted to a correlation analysis between freezing tolerance and tuberization capacity of progenies generated from the first backcrossing with tetraploid cultivars S. tuberosum indicated that these traits are controlled by independent genetic loci (Tu et al., 2021).  (Siri et al, 2009;Santiago et al., 2017). Solanum malmeanum is documented to possess interesting levels of resistance against R. solanacearum phylotype II sequevars 1-2 previously isolated from different potato fields in Uruguay (Siri et al., 2009), evaluated under controlled conditions. Two accessions were classified to be resistant (e.g. absence of wilting symptoms) and three accessions demonstrate moderate resistance. Resistant accessions are summarized in Table 3. Little is known about the resistance across the range of distribution, however, there are ongoing studies aiming at characterizing resistance levels of potato wild species accessions across the distribution in Uruguay. Considering that each strain of bacterial wilt is present at specific geographical regions, the knowledge of its variability and diversity could be an efficient guide to identify new potential sources of resistance from different geographical populations.

Potato late blight
In total thirteen accessions were reported to be resistant to late blight (Table 3). The genotypes OKA 7310.01, OKA 7256.08, OKA 7256.07, OKA 7256.01 showed an incompatible reaction after inoculation with R0 in trials under greenhouse conditions. A second experiment aimed to reconfirm the presence of R-genes and the offspring produced by the cross between OKA 7310.01 (resistant to R0) and OKA 7282.06 (susceptible to R0) was inoculated with a complex race of late blight. Among the offspring of 300 plants, 70% (210) showed incompatible (resistance) responses, confirming the presence of the R genes in S. malmeanum (Micheletto et al., 1999;Micheletto et al., 2000). Micheletto and collaborators (2000) screened four Argentinean diploid accessions for quantitative resistance to late blight Phytophthora infestans virulent (R1, R3, R4, R5, R7, R10, R11) and non-virulent (R0) complex races under both greenhouse and field conditions during two seasons (1996/1997 and 1997/98) to assess the year × genotype interaction. Results showed high variability reaction of P. infestans among genotypes. Two accessions were susceptible (OKA 7282, OKA 7291), while two resistant accessions segregated for resistance (Table 3). OKA 7292 genotypes 5 and 9 and OKA 7256 genotypes 2, 7 and 9 performed as resistant. Furthermore, no genotype × year interaction was detected, and the behavior was consistent across years. Although Micheletto and collaborators (1999;2000) treat the evaluated accessions as S. commersonii, they are currently to be considered as S. malmeanum (Spooner et al., 2014;Spooner et al., 2016;Global Biodiversity Information Facility, 2022). The disease caused by the oomycete Phytophthora infestans (Mont.) de Bary is considered the most important disease of potato crop in all producing regions worldwide (Santana et al., 2013;Göre et al., 2019).

Verticillium wilt
The accession PI 472850 is reported to be immune against Verticillium dahliae (Table 3) (National Plant Germplasm System, USDA, 2022). Verticillium wilt is one of the most important soilborne fungal diseases on potato, also known as potato early dying disease, caused by V. dahliae Klebahn and V. albo-atrum Reinke & Berthold (Frost et al., 2007;Simko and Haynes, 2017;Li et al., 2019).

Fusarium dry rot
One accession (PI 414154) was screened in a tuber disk essay for resistance to Fusarium sambucinum (Lynch et al., 2003), however it responded as susceptible. Fusarium dry rot of potato tubers is caused by many species such as F. avenaceum, F. culmorum, F. equiseti, F. oxysporum, F. sambucinum, F. solani, among others. It is a severe disease responsible for causing significant economic losses mainly during the post-harvest and storage periods (Peters et al., 2008;Du et al., 2012;Gachango et al., 2012).

Nematodes
Solanum malmeanum showed full resistance to potato cyst nematode Globodera rostochiensis and susceptibility to G. pallida, but the inheritance of resistance was not investigated (Castelli et al., 2003). Furthermore, it has also been reported to be resistant against root-knot nematode (Meloidogyne hapla) (Table 3) (National Plant Germplasm System, USDA, 2022). Nematodes, when the population density is high, negatively affect potato production from the field, restricting plant growth, to the post-harvest period, rendering tubers unmarketable (Contina et al., 2019;Koirala et al., 2020).

Insect pests
Solanum malmeanum was moderately resistant against potato leafhopper (Empoasca fabae Harris), although the defense mechanisms have not been elucidated, as well as fully resistant to potato aphid Macrosiphum euphorbiae (Thomas) and potato green peach aphid Myzus persicae (Sulzer) ( Table 3) (Radcliffe and Laufr, 1971;Genesys, 2022;National Plant Germplasm System, USDA, 2022). Insect resistance in potato is mainly associated to morphological characteristics, density on leaves and exudates of secondary metabolites of glandular trichomes, which provide protection against herbivory . Also, an important natural source of insect resistance in potato is related to the production of high levels of foliar glycoalkaloids (Coombs et al., 2002). Some results suggest that this is an important mechanism of insect resistance found in S. malmeanum.

Viruses
Accession PI 458318 from Argentina has been reported to be resistant against potato leaf roll virus (Polerovirus sp.) (Table 3) (Genesys, 2022;National Plant Germplasm System, USDA, 2022). Solanum malmeanum has also been reported to be susceptible and spontaneously infected by the systematic ringspot virus (TMV tobacco mosaic virus) that is transmitted from generation to generation via tubers (Hansen, 1960).
3.6.4 Quality traits for production and industry 3.6.4.1 Tuber dry matter, starch and protein contents Solanum malmeanum has been reported (Table 4) to contain a range from 28.6% to 34.62% of dry matter content (Rocha et al., 2000;Jansen et al., 2001), 15.4 to 23% starch content (Jansen et al., 2001), 4.5% of protein in tuber dry matter content and 32.1% of amylose content in starch, with a mean particle diameter of starch granules of 45.8 mm (CV%=9.2) (Jansen et al., 2001). In addition, reducing sugars on its tubers have been evaluated from two different growing seasons, with an average of 0.22% in spring, and 0.33% in autumn, far below the levels presented in the reference cultivar 'Baronesa' (S. tuberosum) (Chala et al., 2001). A strong association in diploid S. malmeanum clones was verified between glutamate oxaloacetate transaminase (GOT) at relative mobility band 1.00 of electrophoretic standards and high dry matter content (average of 34.62 of dry matter content on tubers). The use of the GOT enzyme may be efficient in more accurately predicting this character in the early stages of selection in diploid than tetraploid species (Andreu and Pereira, 2004).

Glycoalkaloids
Steroidal glycoalkaloids are secondary metabolites toxic for human consumption, although they may also have advantages to improve resistance against viral, insect-pests or microbial diseases. Solanum ser. Commersoniana species mainly produces a-solanine and b-chaconine (Schreiber, 1963). Solanum malmeanum accession PI 320269 (cited as S. commersonii) has been reported to contain 23% (mg/g dry leaves) of glycoalkaloid content on leaves (Table 4), where 100% is dehydrocommersonine, with undetectable levels (<3%) of asolanine and b-chaconine (Gregory et al., 1981) (Table 4). Dehydrocommersonine was associated in Solanum oplocense Hawkes with host resistance against Leptinotarsa decemlineata (Tai et al., 2014;Tai et al., 2015), then S. malmeanum could be predicted as a potential source of resistance against this pest. There is room for selection because they are controlled by genetic factors (Mensinga et al., 2005) and even though high levels of foliar steroidal glycoalkaloids are frequently associated with high contents in tubers (Sanford et al., 1992), they are accumulated during the development of each organ (Krits et al., 2007;Mweetwa et al., 2012;Paudel et al., 2019;Zhen et al., 2019). This differential accumulation behavior allows to change the distribution of accumulation in specific plant tissues across some generations of selection (Sanford et al., 1992;Umemoto et al., 2016;Nahar et al., 2017;Zhen et al., 2019), so it should be possible to select accessions from S. malmeanum for breeding to obtain cultivars with resistance against pests given by high foliar glycoalkaloids content and with low toxicity for human consumption due to low levels of glycoalkaloids in the tubers.
Some accessions of S. malmeanum showed regular meiotic abnormalities observed at microsporogenesis, including chromosomes out of the equatorial plate in metaphases I and II (MI and MII) and lagging chromosomes in anaphases I and II, and telophases I and II (Pandolfi, 1998;Toméet al., 2007;Toméet al., 2009). Furthermore, a range from 3.7% to 50.8% of pollen mother cells (PMC) with meiosis abnormalities was observed. The frequency of abnormal PMC decreased in the later stages of meiosis. This, together with a good pollen viability (60.33 -96.5%), suggests the existence of selection against abnormal PMC cells during the microsporogenesis process with implications for the choice of pollen donors for crosses.
Pollen viability was significantly higher in S. malmeanum than in cultivated potatoes (Pandolfi, 1998;Toméet al., 2007). It also produces a high pollen amount, ranging from 454.20 to 476.20 pollen grains per field, exceeding the means of S. tuberosum clones (Toméet al., 2007). According to Hardigan et al. (2017), the reduction of pollen viability of cultivated potato (S. tuberosum) is a consequence of the domestication syndrome. Although the accessions SCM57 (BGB017) and SCM60 (BGB447) (AleloVegetal, 2022) did not show unreduced pollen grains, they had meiotic mechanisms (parallel spindles) that possibly can lead to the formation of unreduced pollen, presenting the highest percentages of cells with parallel spindles (27% and 22%). Furthermore, pollen grain size of S. commersonii (13.68-35.52 μm) tends to be larger than that of S. malmeanum (11.4 to 23.9 μm) (Toméet al., 2009). This approach put forward by Toméet al., (2009) raises the hypothesis that S. malmeanum has no pollen grain diameters satisfying the value of 25 μm suggested by Quin et al. (1974) and Ramanna (1974) and would imply the proposition of a new threshold for unreduced (2n) pollen grains. However, the selection of 2n pollen based only on diameter and shape of the pollen grain is feasible only in species with known differences between haploid and diploid pollen grains (Parrott and Smith, 1984;Parrott and Smith, 1986). According to Ordoñez et al. (2017), 2n pollen have a diameter 1.2 times larger than reduced gametes (n). 2n gametes production can be predicted by crossing the species of interest with another different EBN group species, and then observing pollen tube growth, fruit set, seed set and germination success. Two of 12 clones of S. malmeanum (PI 414154, cited as "S. commersonii") were reported to produce 2n pollen (den Nijs and Peloquin, 1977a). Therefore, it is expected that in natural populations, functional 2n gametes can be found.

Reproductive biology, breeding system and endosperm balance number
Solanum malmeanum is a tertiary gene pool (1EBN) wild relative of potato and is considered reproductively isolated from other tuberbearing 2EBN and 4EBN species (Hanneman, 1994;Spooner et al., 2016). Solanum malmeanum reproduces vegetatively by tubers and stolons and sexually by botanical seeds. From five diploid accessions, four were characterized as self-incompatible (Nicolao, 2021) and one self-compatible accession of S. malmeanum was found (Nicolao, 2021). Hanneman (1994) used a diploid female plant of S. malmeanum for the assignment of EBN in crossing tests. Although only one accession was used as female to determine its EBN, it produced an average of six seeds per fruit from any crosses with male standard species 2x (1EBN), less than one seed per fruit from 2x (2EBN) species, and no seed set from 4x (4EBN) crosses. Therefore, it was designated as 1EBN.
Crosses between different potato species with the same EBN number, regardless of ploidy, are usually successful (Johnston et al., 1980;Johnston and Hanneman, 1982). Theoretically, species with the same EBN may be intercrossed in nature if sympatry and synchronized flowering occur and pre-zygotic barriers are absent. Tarn and Hawkes (1986) obtained vigorous and fertile hybrid progenies from reciprocal crosses between 2x S. malmeanum and 2x S. commersonii. The seed production per berry and mean weight per 100 seeds were equally variable. Solanum commersonii produces fewer (66.5), but heavier seeds (52.8 mg per 100 seeds) per berry than S. malmeanum (128.4 seeds per berry, with 12.8 mg per 100 seeds) (Tarn and Hawkes, 1986). Furthermore, the seedlings grown from reciprocal crosses between S. commersonii and S. malmeanum usually were vigorous, fertile, highly variable morphologically and generally displayed intermediate morphology between the two parental species (Tarn and Hawkes, 1986). It is interesting to note that parental traits as flower pigmentation (typically purple-pigmented corolla in S. commersonii and white (not-pigmented) corolla in S. malmeanum), always segregate in F1 progenies with intermediate phenotypes (Tarn and Hawkes, 1986). Hawkes and Hjerting (1969) made significant efforts to perform reciprocal crosses with S. chacoense and obtained only one seed that germinated and generated a vigorous plant. Ehlenfeldt and Hanneman (1988) produced a small number of seeds from many hand pollinations between S. malmeanum (accession PI 320269, cited as "S. commersonii") (1EBN) and S. chacoense (2EBN) (Ehlenfeldt and Hanneman, 1988). Obtaining hybrid offspring of S. malmeanum × S. chacoense was possible, but the endosperm barrier between these two species greatly limits viable seed development. In reciprocal backcrosses of the F1 hybrids (1 ½ EBN) with both S. chacoense (2EBN) and S. malmeanum (1EBN), the results were consistent with a directional effect determining the difference in the ratio of viable seeds to aborted seeds, related to which parent is male and which parent is female in any given cross, but it does not seem to be a cytoplasmic effect, because the same female may produce different results depending on pollen source. This effect is most likely due to the dose-effect in the endosperm (Ehlenfeldt and Hanneman, 1988).
Crossability barriers between 2x 2EBN S. chacoense and 2x 1EBN S. malmeanum are well-established, but no sexual barriers were observed between 2x 1EBN S. commersonii and 2x 1EBN S. malmeanum, elucidating that EBN is a strong internal post-zygotic barrier (Johnston et al., 1980;Summers and Grun, 1981;Hanneman, 1994). Thus, it may predict that S. malmeanum could freely cross with other 1EBN sympatric wild potatoes such as 2x 1EBN S. commersonii, but not with 2EBN such as S. chacoense, except when 2n gametes are present (Hawkes and Hjerting, 1969;Hanneman, 1994). Thus, the mechanisms involved in keeping the two species appart in sympatric zones where S. commersonii and S. malmenum occur still deserve further explanations.

Discussion
Solanum malmeanum is a valuable genetic resource for many traits of importance to the potato industry. This 1EBN wild potato contains genes to combat pests and diseases which are lacking in the primary genepool of potato. However, using this 1EBN wild species to improve cultivated potatoes is challenging. S. malmeanum is crossincompatible with cultivated potatoes due to the strong EBN postzygotic barrier (Hanneman, 1994). To capture genetic diversity from 1EBN potato species into the cultivated gene pool, breeders have efficiently used the 2x 2EBN Mexican potato species Solanum verrucosum as bridge species (Jansky and Hamernik, 2009). Bamberg and collaborators (2021) show the ability of female parent S. verrucosum (2EBN) to form interspecific hybrids with 1EBN species as male parent without stylar barrier. Much like S. verrucosum, the lack of stylar barriers in cultivated tomato has allowed tomato breeders to access genetically distant wild relatives, even those of different effective ploidy (Fulton et al., 1997;Roth et al., 2019;Städler et al., 2021). Thus, a combination of strategies such as ploidy manipulation, somatic fusion, embryo rescue and transgenesis are expected not to be required to access the valuable 1EBN potato wild species genetic diversity.
Recently, diploid F1-hybrid breeding has increased attention of breeders due to the possibility of redomestication of potato crop by creating inbred lines from clones carrying mutations of the Sli selfincompatibility gene locus (Lindhout et al., 2011;Jansky et al., 2016;Eggers et al., 2021). Diploid inbred lines are outstanding materials for breeding and functional genetics (Marand et al., 2019;Jayakody et al., 2022), because they allow high throughput phenotyping and efficient QTL detection (Manrique-Carpintero et al., 2015;Hara-Skrzypiec et al., 2018), accelerating the breeding process. An obstacle still present in diploid potato breeding is the high level of inbreeding depression (De Jong and Rowe, 1971;Zhang et al., 2019).
The morphological variability, wide geographic distribution, diverse array of habitats occupied and ecological preferences of S. malmeanum are not fully represented in current genebanks. Limited studies were performed to assess the genetic variability and the few available ones do not fully cover these aspects. Important agronomical traits were documented, although comprehensive studies on this species are lacking and most of the references available for response to abiotic and biotic stresses Micheletto et al., 1999;Micheletto et al., 2000;Castelli et al., 2003) and quality traits for production and industry (Rocha et al., 2000;Chaláet al., 2001;Jansen, 2001) are from occasional works or have evaluated S. malmeanum as "S. commersonii".
Next Generation Sequencing (NGS) technologies will allow a better understanding of the evolutionary relationships between S. malmeanum and S. commersonii to provide the required framework for conserving and using this potato wild genetic diversity (Fielder et al., 2015;Dempewolf et al., 2017). The reinstatement at the species level brings new light to a comeback and promising future for the applied uses of this wild potato in classic and cutting-edge techniques for potato breeding. Towards the true exploitation of this promising potato genetic resource, the current taxonomy resulting from the latest treatment must be updated in all genebanks and reference collections to allow better use of the conserved germplasm and enhance its applied uses. Furthermore, with the rise of new technologies, new studies are needed to evaluate the entire geographical distribution of S. malmeanum and compare to the other two sympatric potato wild species S. chacoense and S. commersonii.
Plant breeders require genetic variability information to speed up the search of each desirable agronomic trait, tolerance against abiotic stresses, as well as for the genes encoding for quality traits (Bethke et al., 2017;Swarup et al., 2021).
As a final remark, we highlight that the potential of S. malmeanum for potato breeding has been neglected, especially when compared to other close potato wild relatives such as S. chacoense and S. commersonii (Aversano et al., 2015;Leisner et al., 2018) and must be properly addressed with contemporary molecular tools to unlock its applied use as a promising potato wild relative to face the challenges imposed to the potato crop in the 21 st century.

Author contributions
RN discussed the original idea, reviewed and checked literature data, compiled and wrote the manuscript. PG wrote partially, provided data on the Uruguayan references, and reviewed the text. CC provided advisory, wrote partially and reviewed the text. GH discussed the original idea, provided advisory, wrote partially and reviewed the text. All authors contributed to the article and approved the submitted version.